Effects of access to feed, water, and a competitive exclusion product in the hatcher on some immune traits and gut development in broiler chickens.

1. This study evaluated the effect of access to feed, water, and the competitive exclusion (CE) product Broilact®, administered in the hatcher, on broiler performance, caecal microbiota development, organ development, intestinal morphology, serum levels of IgY and vaccine-induced antibody responses.2. In total, 250 chicks were hatched in a HatchCareTM hatcher and divided into four groups, given access to feed, water and the CE product sprayed on the chicks (CEs); access to feed, water, and the CE product in water (CEw); access to feed and water (Cpos); or no access to feed and water (Cneg) in the hatcher.3. At the research facility, 10 chicks per hatching treatment were euthanised for organ measurements. The remaining 200 chicks were randomly distributed to 20 pens. On d 11, all birds were vaccinated against avian pneumovirus (APV). Three focal birds per pen were blood-sampled weekly for quantification of IgY and serum antibodies to APV. On d 11 and 32, two birds per replicate pen were euthanised for organ measurements and sample collection. Feed intake and body weight were recorded weekly.4. Delayed access to feed and water reduced weight gain and feed intake early in life. At the end of the study, no differences in body weight remained.5. There were some early effects on organs, with depressed intestinal development and higher relative gizzard weight for the Cneg group at placement. No treatment effects on the immune traits measured were detected.6. The relative abundance of seven bacterial genera differed between treatment groups at d 11 of age. The results suggested that chickens are capable of compensating for 40 h feed and water deprival post-hatch. Provision of Broilact® did not have any persistent performance-enhancing properties, although different outcomes under rearing conditions closer to commercial production cannot be ruled out.

Monitoring of local CD8ß-expressing cell populations during Eimeria tenella infection of naïve and immune chickens.

The purpose of this study was to monitor abundance and activation of local CD8ß-expressing T-cell populations during Eimeria tenella infections of naïve chickens and chickens immune by previous infections. Chickens were infected with E. tenella up to three times. Caecal T-cell receptor (TCR) γ/δ-CD8ß+ cells (cytotoxic T lymphocytes; CTL) and TCRγ/δ+CD8ß+ cells were characterized with respect to activation markers (blast transformation, CD25 and cell surface CD107a). Cells were also induced to degranulate in vitro as a measure of activation potential. Major findings included a prominent long-lasting, up to 6 weeks, increase in the proportion of CTL among caecal CD45+ cells in the later stages after primary E. tenella infection. These CTL also showed clear signs of activation, that is blast transformation and increased in vitro induced degranulation. At second and third E. tenella infection, chickens showed strong protective immunity but discrete signs of cellular activation were observed, for example increased in vitro induced degranulation of CTL. Thus, primary E. tenella infection induced clear recruitment and activation of local CTL. Upon subsequent infections of strongly immune chickens cellular changes were less prominent, possibly due to lower overall numbers of cells being activated because of the severe restriction of parasite replication.

Expression of perforin, granzyme A and Fas ligand mRNA in caecal tissues upon Eimeria tenella infection of naïve and immune chickens.

Cytotoxic cells of the immune system may kill infected or transformed host cells via the perforin/granzyme or the Fas ligand (FasL) pathways. The purpose of this study was to determine mRNA expression of perforin, granzyme A and FasL in Eimeria tenella-infected tissues at primary infection and infection of immune chickens as an indirect measure of cytotoxic cell activity. Chickens were rendered immune by repeated E. tenella infections, which were manifested as an absence of clinical signs or pathological lesions and significantly reduced oocyst production upon challenge infection. During primary E. tenella infection, perforin, granzyme A and FasL mRNA expression in caecal tissue was significantly increased at 10 days after infection, compared to uninfected birds. In contrast, at infection of immune birds, perforin and granzyme A mRNA expression in caecal tissue was significantly increased during the early stages of E. tenella challenge infection, days 1-4, which coincided with a substantial reduction of parasite replication in these birds. These results indicate the activation of cytotoxic pathways in immune birds and support a role for cytotoxic T cells in the protection against Eimeria infections.

Cytokine mRNA expression in bronchoalveolar lavage cells during Dictyocaulus viviparus infection in calves.

The purpose of this study was to monitor local cytokine responses to Dictyocaulus viviparus in calves during primary infection and re-infection. Bronchoalveolar lavage fluid (BALF) was collected weekly from experimentally infected calves and interleukin-2 (IL-2), IL-4, IL-5, IL-10 and IFN-γ mRNA expression was quantified in BALF cells. The major finding was a prominent transient increase in IL-4 mRNA expression, compared with that of uninfected calves, observed in BALF cells collected 2-3 weeks post-primary D. viviparus infection. At 2 weeks post-infection, macroscopic worms were also first observed in BALF. Calves re-infected after 10 weeks were partially immune which was evident at slaughter 5 weeks post-infection as a lower worm burden than in previously naïve calves infected at the same time. IL-4 mRNA expression in BALF cells 2 weeks post-re-infection was increased compared with that of uninfected animals but not as high as that of primarily infected calves. BALF cell expression of the other cytokines tested for was not as clearly effected by the D. viviparus infection. It seems likely that the strong IL-4 response observed during primary infection reflects an innate response to the worms that may initiate an ensuing Th2 response, which confers protective immunity.

Effects of algal supplementation in feed to broiler breeders on transfer of nutrients and antibodies to chicks and quality of hatchlings.

Breeder nutrition is an important factor for chick quality since the chick embryo relies on nutrients available in the egg for growth and development. In addition, the egg is providing the chick with important antibodies that are vital during the first weeks of life. Brown algae contains several bioactive compounds, and dietary supplementation with algal extracts have shown improved gut health and immune responses in both pigs and poultry. The aim of this study was to investigate if feeding the brown algae Saccharina latissima, intact or as an extract, to broiler breeders can affect breeder hens' antibody responses to vaccination, egg quality and transfer of antibodies and nutrients to the egg and thereby improve the quality of newly hatched chicks. Forty-five hens and nine roosters of the parent lines of the fast-growing broiler Ross 308 were included in the experiment where hens were 31 weeks at the start. The hens were housed individually and fed one of three dietary treatments for seven weeks; (a) control, (b) addition of 0.6% algal meal or (c) addition of 0.08% algal extract. The hens were given a booster vaccination against infectious bronchitis virus (IBV) 21 days after the start of experiment. During experimental days 32-42, hens were naturally mated every 5th day and hatching eggs were collected. A total of 255 chicks were hatched, and chick quality was assessed. Moreover, on chick day three, blood was collected from 48 focal chickens and total immunoglobulin Y levels and specific titres to IBV in serum were determined. The results showed that feeding the brown algae Saccharina latissima, intact or as an extract to broiler breeders did not affect egg production, egg quality, antibody responses to vaccination or transfer of antibodies from hen to chick. However, feeding intact algae significantly increased the levels of iodine and decreased the level of selenium in the eggs and resulted in a lower proportion of chicks with maximum quality score. Interestingly, algal feeding, both intact and as an extract, increased the abdominal fat pad in broiler breeders by about 17% without affecting BW. In conclusion, supplementation of broiler breeder diets with algal extract from Saccharina latissima, but not intact algal meal is a promising dietary strategy to increase the abdominal fat pad without causing any adverse effects on nutrient level in eggs or chick quality.

Characterization of bovine lymphocytes stimulated in vitro by Dictyocaulus viviparus homogenate.

Adult Dictyocaulus viviparus homogenate induced proliferation of lymphocytes from naïve cattle. We characterized the responding cells by carboxyfluorescein diacetate succinimidyl ester (CFSE) loading, for detection of proliferation, and antibody labelling for cell surface molecules. Lymphocytes expressing CD4, CD8 and gamma/delta TCR, rather than Ig expressing cells, proliferated after in vitro stimulation with D. viviparus homogenate. Of gamma/delta TCR expressing cells, both CD8, WC1.1 and WC1.2 co-expressing cells proliferated. Moreover, gamma/delta T cells expressing MHC class II proliferated to a higher extent than those negative for MHC class II. Of CD4 and CD8 expressing lymphocytes, both those co-expressing CD45R and CD45R0 proliferated. Among CD4 expressing lymphocytes, those that were CD45R0 positive had a larger proportion of proliferated cells than did CD45R positive cells. Compared to stimulation with Con A, the proportion of dividing cells after D. viviparus stimulation was smaller although the cells had divided more times. Furthermore, we also compared in vitro responses of peripheral blood mononuclear cells collected before and after two subsequent infections with D. viviparus, but no clear acquired responses could be detected. Overall, this suggests that most T lymphocytes are stimulated by the D. viviparus homogenate rather than any particular lymphocyte subpopulation.

Quantification and phenotypic characterisation of peripheral IFN-γ producing leucocytes in chickens vaccinated against Newcastle disease.

The aim of this study was to optimise and evaluate an intracellular cytokine staining (ICS) assay for assessment of T cell IFN-γ responses in chickens vaccinated against Newcastle disease (ND). We aimed to validate currently available antibodies to chicken IFN-γ using transfected CHO cells. Moreover, this ICS assay was evaluated for use to detect mitogen and antigen induced IFN-γ production in chicken peripheral blood leucocytes. Chickens from an inbred white leghorn line containing two MHC haplotypes, B19 and B21, were divided into three experimental groups; one group was kept as naive controls, one group was vaccinated intramuscularly twice with a commercial inactivated ND virus (NDV) vaccine, and the last group was vaccinated orally twice with a commercial live attenuated NDV vaccine. PBMC were ex vivo stimulated with ConA or with NDV antigen. The ICS assay was used to determine the phenotype and frequency of IFN-γ positive cells. ConA stimulation induced extensive IFN-γ production in both CD3+TCRγδ+ (γδ T cells) cells and CD3+TCRγδ- cells (αß T cells), but no significant differences were observed between the experimental groups. Furthermore, a large proportion of the IFN-γ producing cells were CD3- indicating that other cells than classic T cells, secreted this cytokine. NDV antigen stimulation induced IFN-γ production but to a lower extent than ConA and with a large variation between individuals. The CD3+TCR1γδ-CD8α+ (CTL) population produced the highest NDV specific IFN-γ responses, with significantly elevated levels of IFN-γ producing cells in the B19 chickens vaccinated orally with live attenuated NDV vaccine. This was not the case in the B21 animals, indicating a haplotype restricted variation. In contrast, the CD3+TCR1γδ-CD4+ (Th) population did not show a significant increase in IFN-γ production in NDV stimulated samples which was in part due to a high number of IFN-γ producing cells after incubation with medium alone. In conclusion, an ICS assay for phenotyping of IFN-γ producing chicken leukocytes was set up that proved useful in identifying cytokine producing cells upon either mitogen or antigen-specific stimulation.

Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2.

An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-alpha in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.

Lectins inhibit the Aujeszky's disease virus-induced interferon-alpha production of porcine peripheral blood mononuclear cells.

The interaction between virus and peripheral blood mononuclear cells (PBMC) required to elicit the production of interferon-alpha (IFN-alpha) by the so-called natural interferon-producing cell is unknown. However, results from inhibition experiments suggest that viral glycoproteins are essential in this IFN induction process. We demonstrate here that cellular glycoproteins also appear to be involved in the initiation of IFN-alpha production. Lectins, that is, sugar binding glycoproteins, inhibited the Aujeszky's disease virus-induced IFN-alpha production of porcine PBMC by up to 99%. The level of inhibition varied with lectin used (concanavalin A, Galanthus nivalis lectin, Helix pomatia lectin, and lentil lectin). Preincubation experiments with porcine cells and concanavalin A (ConA) revealed that the lectin exerted its major effect directly on the PBMC. Although the IFN-alpha production in some cases was reduced by more than 90%, the PBMC were still able to proliferate in response to mitogenic lectins. The ConA-mediated inhibition of the IFN-alpha production was reduced if the lectin was added later than 6-8 h after the start of induction and was not mediated by soluble factors. Both orthovanadate and staurosporine inhibited the IFN-alpha production and did not relieve the ConA-mediated inhibition. Thus, ConA seems to interfere with the early events during IFN-alpha induction, but the mechanisms behind this interference could not be clarified.

Actinobacillus pleuropneumonia serotype 2--effects on the interferon-alpha production of porcine leukocytes in vivo and in vitro.

Effects of a bacterial infection on the IFN-alpha production in vivo and in vitro were studied in eight specific pathogen free pigs experimentally infected with Actinobacillus pleuropneumoniae. Clinically, the experimental infection was manifested as a febrile stage which lasted approximately one week and by signs of respiratory disease. The Aujeszky's disease virus (ADV) induced IFN-alpha production, assessed in whole blood cultures, was increased for the infected pigs during the febrile stage. Potentiating effects on the IFN-alpha production could be transferred to cultures of purified peripheral blood mononuclear cells with sera collected from the infected pigs during this period of time. Although the experimental infection with A. pleuropneumoniae did not induce any detectable amounts of IFN-alpha in serum or nasal secretion, both a phenol-extract and a heat-inactivated preparation of the bacteria induced low levels of IFN-alpha in cultures of purified PBMC. The interferogenic structures of the bacteria were not identified but there were indications that the bacteria induced IFN-alpha production in the same cell type as ADV.

Pre-infection frequencies of equine herpesvirus-1 specific, cytotoxic T lymphocytes correlate with protection against abortion following experimental infection of pregnant mares.

In general, vaccines containing inactivated equine herpesvirus-1 (EHV-1) fail to prevent abortion in pregnant mares following infection with a virulent strain of EHV-1. We have tested the hypothesis that resistance to EHV-1-induced abortion in pregnant mares is associated with high frequencies of EHV-1 specific, major histocompatibility complex (MHC) class I-restricted, cytotoxic T lymphocytes (CTL) in the circulation. To test this theory, three groups of pregnant mares were assembled with varying backgrounds of infection or vaccination in an attempt to mimic the immune status of the general population. Group 1 mares (n=9) were untreated controls selected at random. Group 2 mares (n=5) were vaccinated three times intramuscularly with inactivated EHV-1. Group 3 mares (n=3) had been infected with EHV-1 on four previous occasions. The frequency of CTL in blood leucocytes was measured by limiting dilution analysis at three time points; at the beginning of pregnancy (approximately 28 weeks before infection) in the Group 2 and Group 3 mares (4-7 weeks of gestation) (Group 1 was unavailable for sampling) and then 2 weeks before (30-40 weeks of gestation) and 3 weeks after experimental infection in all the mares. Serum samples were collected to monitor complement fixing (CF) antibody titres. Mares in all three groups were infected experimentally with EHV-1 strain Ab4/8 by the intranasal route after which they were monitored clinically to determine the outcome of pregnancy and samples were collected to determine the duration of nasopharyngeal shedding and cell-associated viraemia. The untreated control mares showed low pre-infection CTL. After experimental infection, they all seroconverted, aborted and demonstrated expected clinical and virological signs. Some vaccinated mares (3/5) had elevated titres of CF antibody prior to their first vaccination. All the vaccinated mares seroconverted after vaccination and exhibited higher CTL frequencies than controls before infection. Four of the five foaled normally. The multiply infected mares had low CF antibody titres prior to infection and showed neither seroconversion nor clinical or virological signs after infection. All multiply infected mares exhibited high frequencies of CTL before infection and they all foaled normally. The CTL frequencies observed differed significantly from the expected frequencies in the control and multiply infected groups at 2 weeks pre-infection (P=0.034) and between the foaling and aborting mares at 2 weeks pre-infection (P=0.005) and 3 weeks post-infection (P=0.015). The results show a positive correlation between the number of virus-specific CTL in the peripheral blood of pregnant mares and their protection against abortion induced by EHV-1 infection. Therefore, as indicated by this study, rational approaches to the development of new vaccines for EHV-1 should stimulate cytotoxic immune responses and develop virus-specific CTL as pre-requisites for protection against abortion.

Tissue chambers--a useful model for in vivo studies of cytokine production in the pig.

An in vivo tissue chamber model was developed to enable studies of local cytokine production and cellular events during inflammatory and immune reactions in the pig. Tissue chambers made of sialistic rubber tubing were surgically implanted in the subcutaneous tissue- and samples of tissue chamber fluid (TCF) and inflammatory cells were collected by aspiration with a syringe. To evaluate the model for local cytokine production, two cytokine inducers, polyribinosinic-polyribocytidylic acid (poly I:C) and fixed Aujeszky's disease virus infected PK15 cells (ADV-PK15), were injected into the tissue chambers and samples of TCF were collected 0, 4, 8, 12, 24 and 48 h post injection. Poly I:C injections induced local production of interferon-alpha (IFN-alpha) as well as tumor necrosis factor (TNF) in the TCF but kinetic differences in the production of the cytokines were noted. Poly I:C also induced an increase in cell numbers in the TCF, mainly due to increased neutrophil numbers. Injections of ADV-PK15 induced local IFN-alpha production in the TCF as long as the pigs were serologically negative to ADV. Immunofluorescence and in situ hybridization techniques could be applied for characterization of TCF cells. Moreover, cells recovered from the tissue chambers were viable and could be used in functional in vitro tests. Taken together, this tissue chamber model could prove very useful in in vivo studies of inflammatory/immune responses and cytokine production in the pig.

Evaluation of various cytokines (IL-6, IFN-alpha, IFN-gamma, TNF-alpha) as markers for acute bacterial infection in swine--a possible role for serum interleukin-6.

A total of 64 specific pathogen free pigs were divided into eight experimental groups. Pigs in Group I served as non-infected controls while the other 56 pigs were infected intranasally with approximately 7 x 10(8) CFU of Actinobacillus pleuropneumoniae serotype 2 (strain 700/89) in 1 ml saline. When more than 25% of the infected animals showed clinical signs of disease, i.e. 20 h post infection, 48 of the infected pigs were treated with different antibiotics (8 pigs per group), leaving 8 infected animals untreated. Serum samples collected 0, 10, 20, 28 and 44 h, and 3, 4, 7, 13 and 17 days post infection were analysed for their content of interferon (IFN)-alpha, IFN-gamma, tumor necrosis factor (TNF)-alpha by immunoassays and interleukin-6 (IL-6) by a bioassay. In addition, the development of specific antibodies was determined in sera. Among the cytokines analysed, the experimental infection only induced detectable serum levels of IL-6. The appearance of IL-6 positive animals coincided with the onset of clinical signs of disease and increased body temperatures. Varying levels of IL-6 (range, 1-220 U ml-1) were detected in serum from a majority of the infected pigs (80%). In general, the highest levels of IL-6 were detected in serum collected for 10 or 20 h after infection. Among the animals not treated with antibiotics, the number of pigs displaying IL-6 in serum continued to increase until 28 h post infection and then declined. The duration of the IL-6 response varied between individuals and lasted from eight hours to three days. Treatment with antibiotics that ceased the infection also terminated the IL-6 production in most of the pigs. In a pilot field survey, IL-6 was detected in an approximately 30% of serum samples collected from conventional reared pigs before allocation to finishing units. Thus, serum IL-6 seems to be a potential marker for ongoing bacterial infections in swine.

Genetic variation in parameters reflecting immune competence of swine.

Genetic variation in total and differential white blood cell (WBC) counts, phagocytic capacity of polymorphonuclear leukocytes (PMNL), virus induced interferon-alpha (IFN-alpha) production, mitogen induced proliferation and interleukin 2 (IL-2) production of mononuclear cells (MNC) in vitro was studied in blood collected from 124 Yorkshire piglets, aged 8 weeks. The piglets were the offspring from 12 sires and 31 dams. Data from an earlier experiment, including 96 piglets of seven sires and 24 dams, were added when estimating heritabilities for Con A induced proliferation and IL-2 production. The highest heritability (h2 = 0.87 +/- 0.41) was estimated for the total number of PMNL. Medium high heritabilities (h2 = 0.3-0.4) were estimated for the phagocytic capacity of PMNL, Con A induced proliferation and IL-2 production and the total number of WBC, while the heritability estimates were lower (h2 = 0.00-0.08 +/- 0.12) for the total number of lymphocytes, serum concentrations of Ig and IFN-alpha production. Pronounced differences between litters from various dams were found for total number of lymphocytes, IFN-alpha production, Con A induced proliferation and IL-2 production. The Con A induced proliferation was positively correlated (r = 0.48, P < 0.001) with the IL-2 production and both these parameters were correlated (r = 0.44 and 0.37, respectively, P < 0.001) to the virus induced IFN-alpha production. Despite these positive correlations, no parental offspring group was uniformly superior across all traits measured. However, the heritabilities estimated for the immune parameters are sufficiently high to be used as genetic markers in selection for general immune competence of swine.

Determination of equid herpesvirus 1-specific, CD8+, cytotoxic T lymphocyte precursor frequencies in ponies.

The frequency of antigen-specific, genetically restricted cytotoxic T lymphocyte precursors (CTLp) was measured in peripheral blood mononuclear cells (PBMC) of ponies before and after infection with equid herpesvirus 1 (EHV1). Split-well limiting dilution analysis (LDA) was developed to measure CTLp frequency using EHV1-infected 51Cr-labelled lymphoblasts as targets. Extensive characterisation showed that recombinant human interleukin-2, autologous antigen presenting cells and equine serum containing virus neutralising antibody were necessary for maturation of CTLp into effector CTL in vitro. CTLs were not induced when the equine serum (containing VN antibody) was replaced with either foetal calf serum or foetal equine serum (without VN antibody), or seronegative equine serum. CTLp frequency decreased significantly when CD8+ lymphocytes were depleted from the induction cultures. There was good inter- and intra-assay reproducibility using both fresh and recovered cryopreserved PBMC. Both EHV1 and EHV4 could be used to induce effector CTL which lysed EHV1-infected target cells. CTLp frequencies were measured in 2 groups of ponies: Group 1 consisted of two ponies (approx. 9 years old), which had multiple previous experimental infections with EHV1; Group 2 comprised five young (1-2 years) and two older (7 years) ponies which had presumed natural exposure to EHV1/EHV4 but no previous experimental infections. The results showed that CTLp frequencies were higher in the ponies of Group 1 compared with the others. Moreover, ponies with the higher CTLp frequencies were better protected against re-challenge infection with EHV1, showing reduced or absent clinical and virological signs. Consequently, measurement of EHV1-specific CTLp frequency is a potential in vitro correlate of immunity which may be useful for screening new vaccines in horses before embarking upon challenge protection studies to confirm efficacy.

Toxoplasma gondii infection in the mountain hare (Lepus timidus) and domestic rabbit (Oryctolagus cuniculus). II. Early immune reactions.

As already reported, the mountain hare is much more susceptible than the domestic rabbit to oral inoculation with Toxoplasma gondii, as judged by pathological changes and dissemination of parasites within the body. In the present paper, further interspecies variations are reported. Concentrations of the acute phase reactant haptoglobin were raised in hares but not in rabbits one week post-infection (pi), probably reflecting the severe tissue damage present. No difference in the early humoral immune response of hares and rabbits was found, both species producing IgM and IgG antibodies to T. gondii one week pi. Lymphocyte stimulation tests performed before and one week after inoculation showed a high proliferative response to the parasite in blood cell cultures from rabbits but not hares. The fatal outcome of T. gondii infection in the hares is probably due, at least in part, to the lack of cellular response.

Effects of stress resulting from short-term restraint on in vitro functional capacity of leukocytes obtained from pigs.

OBJECTIVE: To investigate whether the procedure used to snare and restrain pigs during collection of blood samples would alter in vitro functional capacity of leukocytes in the samples. ANIMALS: 8 gilts. PROCEDURE: Catheters were surgically inserted into the jugular vein of gilts to enable blood sample collection without restraint. After collection of a control sample, gilts were restrained by use of a snare and samples were collected at 0.5, 3.5, and 6.5 minutes after start of restraint (0 minutes). At each time point, plasma beta-endorphin and cortisol concentrations as well as WBC counts were recorded, and functional capacity of leukocytes in cultures of whole blood was assessed by means of mitogen-induced proliferation and interleukin-2 activity, virus-induced interferon-alpha concentration, and phagocytosis of zymosan particles. RESULTS: Concentrations of plasma beta-endorphin and cortisol were increased at 3.5 and 6.5 minutes after start of restraint. At these times, virus-induced interferon-alpha concentration was decreased, whereas proliferative response to Concanavalin A and phytohemagglutinin increased in samples collected at 6.5 minutes. CONCLUSION AND CLINICAL RELEVANCE: It was possible to snare pigs for the purpose of collecting blood samples and restrain them without causing excessive stress that would affect immunologic variables, provided that the collection procedure was completed within a few minutes.

Genetic and stress-mediated influence on Aujeszky's disease virus induced interferon-alpha production in porcine leukocytes.

The ability to produce interferon-alpha (IFN-alpha) in vitro was measured in blood from 200 F2-crosses between European wild boar and Swedish Yorkshire pigs, originating from a reference pedigree for gene mapping. A total of 200 pigs of 44 litters, descendent from 4 boars and 22 sows, were stressed by transportation together with non-littermates for 5 h. Blood samples were collected from each individual twice, i.e. immediately before transportation and the day after transportation. IFN-alpha production was induced in whole blood cultures by a monolayer of fixed, Aujeszky's disease virus infected, porcine kidney cells. In general, the amount of IFN-alpha produced was significantly lower (p = 0.02) the day after transportation, although the ability to produce IFN-alpha showed a large individual variation (p < 0.001). However, both the levels of IFN-alpha produced and the decrease after transportation varied between the four parental offspring groups. Also, indications of single genes with significant effects on the ability to produce IFN-alpha were found. These results confirm a genetic influence on the ability to produce IFN-alpha. In addition, stress, such as transportation and mixing, may decrease the level of IFN-alpha produced.

Flow cytometric assessment of antigen-specific proliferation in peripheral chicken T cells by CFSE dilution.

Carboxyfluorescein succinimidyl ester (CFSE) dilution is a well established method for analysis of dividing cells by flow cytometry. In other species the method has been extensively used in the study of antigen-specific T cells. The purpose of this study was to apply the method to chicken peripheral mononuclear blood cells (PBMC) and to evaluate and optimize its performance in relation to detection of vaccine-induced chicken T cells specific for Newcastle disease virus (NDV). The method was based on analysis of CFSE dilution upon ex vivo recall stimulation with whole vaccine antigen. Analysis of proliferation was combined with the use of monoclonal antibodies directed against the lymphocyte surface markers CD4 and CD8 in order to phenotype the responding cells. Problems with nonspecific background proliferation especially in the CD8 compartment were significantly reduced by replacing medium containing fetal calf serum with serum-free medium. It was rendered probable that antigen-specific cellular immunity can be assessed by this method as NDV-vaccinated chickens showed a significantly higher proliferative capacity than age-matched naïve controls. Furthermore it was shown that the recall stimulation lead to a proliferative response in T cells expressing αß-type TCRs but also those expressing the γδ-type. In summary, the method was found challenging but nevertheless useful to quantify the proliferative response of chicken antigen-specific T cells. Further investigations though, are needed in order to prove what cell subsets are true antigen-specific responders and what cells are bystander activated. Nevertheless, the method is expected to be a valuable tool to evaluate and quantify vaccine responses to current and new chicken vaccines in the future.